The stereoselective reduction of an α-halo-ketone to its corresponding chiral halo-alcohol is a transformation found in many useful synthetic routes. For example, a synthetic route to the antiviral compound, atazanavir, involves the reduction of a Boc-chloro-ketone derived from L-phenylalanine to the corresponding chiral Boc-(S)-chloro-alcohol. Standard chemical techniques for carrying out this transformation result in diastereomeric mixtures of the desired intermediate that require further resolution, increasing cost and lowering efficiency in the production of atazanavir. Accordingly, processes and compositions capable of more efficient stereoselective reductions of α-halo-ketones to chiral halo-alcohols would be desirable.
Certain enzymes belonging to the ketoreductase (KRED) or carbonyl reductase class (EC1.1.1.184) have been found to be useful for the stereoselective conversion of pro-stereoisomeric aldehyde or ketone substrates to the corresponding chiral alcohol products. KREDs typically convert a ketone or aldehyde substrate to the corresponding alcohol product, but may also catalyze the reverse reaction, oxidation of an alcohol substrate to the corresponding ketone/aldehyde product. The reduction of ketones and aldehydes and the oxidation of alcohols by enzymes such as KRED requires a co-factor, most commonly reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH), and nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) for the oxidation reaction. NADH and NADPH serve as electron donors, while NAD and NADP serve as electron acceptors.
KREDs are increasingly being used for the stereoselective conversion of ketones and aldehydes to chiral alcohols compounds used in the production of key pharmaceutical compounds. Examples using KREDs to generate useful chemical compounds include asymmetric reduction of 4-chloroacetoacetate esters (Zhou, J. Am. Chem. Soc. 1983 105:5925-5926; Santaniello, J. Chem. Res. (S) 1984:132-133; U.S. Pat. Nos. 5,559,030; 5,700,670 and 5,891,685), reduction of dioxocarboxylic acids (e.g., U.S. Pat. No. 6,399,339), reduction of tert-butyl(S)chloro-5-hydroxy-3-oxohexanoate (e.g., U.S. Pat. No. 6,645,746 and WO 01/40450), reduction of pyrrolotriazine-based compounds (e.g., U.S. application No. 2006/0286646); reduction of substituted acetophenones (e.g., U.S. Pat. No. 6,800,477); and reduction of ketothiolanes (WO 2005/054491). In another approach, as demonstrated herein, the ketoreduction can be carried out in the presence of an alcohol, such as isopropanol, to provide a substrate for the reverse reaction (alcohol dehydrogenation). In this manner, the NADH/NADPH consumed in the ketoreduction reaction is regenerated by the reverse, oxidative reaction.
U.S. Pat. No. 7,083,973 discloses a stereoselective process for the preparation of (3S,2R)-1-halo-2-hydroxy-3-(protected)amino-4-substituted butanes by the reduction of the corresponding keto group containing compounds using certain species of Rhodococcus and Brevibacterium. The '973 patent discloses that only selected species of Rhodococcus and Brevibacterium catalyze the reduction to form the desired (3S,2R)-1-halo-2-hydroxy-3-(protected)amino-4-substituted butanes in high quantitative and enantiomeric yield. The '973 patent discloses that 10 mL of cell extract from 150 g of Rhodococcus erythropolis ATCC 4277 cells loaded with 10 mg of (1S)-[N-(1-benzyl-2-oxo-3-chloro)propyl]carbamic acid t-butyl ester substrate, glucose dehydrogenase (35 units), 0.7 mM NAD+ and 200 mg of glucose (reaction carried out at pH 6.0, 150 RPM agitation and 30° C.) results in (1S,2R)-[N-(1-benzyl-2-hydroxy-3-chloro)propyl]carbamic acid t-butyl ester product in 95% yield with >98% diastereomeric purity.
Accordingly, isolated KRED polypeptides capable of stereoselective conversion of α-halo-ketones to chiral halo-alcohols in high-yield and high diastereomeric purity would be desirable. Also, improved processes for using KRED polypeptides to carry out large-scale preparation of chiral halo-alcohols would be desirable.